Todar's Online Textbook of Bacteriology

THE CONTROL OF MICROBIAL GROWTH

2002 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology

Introduction The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology. "Control of growth", as used here, means to prevent growth of microorganisms. This control is effected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of microorganisms. Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without killing them) are referred to as static agents. Thus the term bactericidal refers to killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria, a fungicide kills fungi, and so on. Sterilization is the complete destruction or elimination of all viable organisms (in or on an object being sterilized). There are no degrees of sterilization: an object is either sterile or not. Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of cells. Methods of Sterilization Heat: most important and widely used. For sterilization always consider type of heat, time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.

Incineration: burns organisms and physically destroys them. Used for needles , inoculating wires, glassware, etc. and objects not destroyed in the incineration process. Boiling: 100o for 30 minutes. Kills everything except some endospores (Actually, for the purposes of purifying drinking water 100o for five minutes is probably adequate though there have been some reports that Giardia cysts can survive this process). To kill endospores, and therefore sterilize the solution, very long or intermittent boiling is required. Autoclaving (steam under pressure or pressure cooker) : 121o for 15 minutes (15#/in2 pressure). Good for sterilizing almost anything, but heat-labile substances will be denatured or destroyed. Dry heat (hot air oven): 160o/2hours or 170o/1hour. Used for glassware, metal, and objects that won't melt. The protocol and recommendations for the use of heat to control microbial growth are given in Table 1. Table 1. Recommended use of heat to control bacterial growth Treatment Temperature Effectiveness Vaporizes organic material on nonflammable surfaces but may Incineration >500o destroy many substances in the process 30 minutes of boiling kills microbial pathogens and vegetative forms of Boiling 100o bacteria but may not kill bacterial endospores Three 30-minute intervals of boiling, o Intermittent boiling 100 followed by periods of cooling kills bacterial endospores kills all forms of life including Autoclave and o 121 /15 bacterial endospores. The pressure cooker minutes at substance being sterilized must be (steam under 15# pressure maintained at the effective T for the pressure) full time For materials that must remain dry and which are not destroyed at T Dry heat (hot air 160o/2 hours between 121o and 170o Good for oven) glassware, metal, not plastic or rubber items o Dry heat (hot air 170 /1 hour Same as above. Note increasing T oven) by 10 degrees shortens the

Pasteurization (batch method)

Pasteurization (flash method)

sterilizing time by 50 percent kills most vegetative bacterial cells including pathogens such as 63o/30 minutes streptococci, staphylococci and Mycobacterium tuberculosis Effect on bacterial cells similar to batch method; for milk, this method o 72 /15 seconds is more conducive to industry and has fewer undesirable effects on quality or taste

Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is usually used (commonly used to sterilize the surfaces of objects), although x-rays and microwaves are possibly useful. Filtration: involvres the physical removal (exclusion) of all cells in a liquid or gas, especially important to sterilize solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.) Chemical and gas: (formaldehyde, glutaraldehyde, ethylene oxide) toxic chemicals kill all forms of life in a specialized gas chamber. Control of Microbial Growth by Physical Agents Applications of Heat The lethal temperature varies in microorganisms. The time required to kill depends on the number of organisms, species, nature of the product being heated, pH, and temperature. Whenever heat is used to control microbial growth inevitably both time and temperature are considered. Sterilization (boiling, autoclaving, hot air oven) kills all microorganisms with heat; commonly employed in canning, bottling, and other sterile packaging procedures. Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or food. In the case of pasteurization of milk the time and temperature depend on killing potential pathogens that are transmitted in milk, i.e., staphylococci, streptococci, Brucella abortus and Mycobacterium tuberculosis. For pasteurzation of milk: batch nethod: 63o/30minutes; flash method: 71o/15 seconds. Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at 0o. Store perishable foods at low

temperatues to slow rate of growth and consequent spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs, rather than true psychrophiles, are the usual cause of food spoilage in refrigerated foods. Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity (Aw < 0.90). Often used to preserve foods (e.g. fruits, grains, etc.). Methods involve removal of water from product by heat, evaporation, freeze-drying, addition of salt or sugar. Irradiation (microwave, UV, x-ray): destroys microorganisms as described under "sterilization". Many spoilage organisms are easily killed by irradiation. In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent. The practice has not been accepted in the U.S. Control of microbial growth by chemical agents Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial agents include chemical preservatives and antiseptics, as well as drugs used in the treatment of infectious diseases of plants and animals. Antimicrobial agents may be of natural or synthetic origin, and they may have a static or cidal effect on microorganisms. Types of antimicrobial agents Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane; should not be taken internaslly. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents. Disinfectants: Agents that kill microorganisms, but not necessarily their spores,not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, lye, copper sulfate, quaternary ammonium compounds. Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink.

Common antiseptics and disinfectants and their uses are summarized in Table 2.
Table 2. Common antiseptics and disinfectants

Chemical

Action Denatures proteins Ethanol (50-70%) and solubilizes lipids Denatures proteins Isopropanol (50-70%) and solubilizes lipids Reacts with NH2, Formaldehyde (8%) SH and COOH groups Tincture of Iodine (2% I2 in Inactivates 70% alcohol) proteins Forms hypochlorous acid Chlorine (Cl2) gas (HClO), a strong oxidizing agent Silver nitrate (AgNO3) Precipitates proteins Inactivates proteins by reacting with sulfide groups Disrupts cell membranes

Uses Antiseptic used on skin Antiseptic used on skin Disinfectant, kills endospores Antiseptic used on skin Disinfect drinking water; general disinfectant General antiseptic and used in the eyes of newborns Disinfectant, although occasionally used as an antiseptic on skin Skin antiseptics and disinfectants

Mercuric chloride Detergents (e.g. quaternary ammonium compounds) Phenolic compounds (e.g. carboloic acid, lysol, hexylresorcinol, hexachlorophene) Ethylene oxide gas

Antiseptics at low Denature proteins concentrations; and disrupt cell disinfectants at high membranes concentrations Disinfectant used to sterilize heat-sensitive Alkylating agent objects such as rubber and plastics

Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If eaten they should be nontoxic. Examples; calcium propionate, sodium benzoate, formaldehyde, nitrate, sulfur dioxide. Table 3 is a list of common preservative and their uses.

Table 3. Common food preservatives and their uses

Preservative Propionic acid and propionates Sorbic acid and sorbates Benzoic acid and benzoates Sodium diacetate Lactic acid Sulfur dioxide, sulfites Sodium nitrite Sodium chloride Sugar Wood smoke

Effective Concentration 0.32% 0.2% 0.1% 0.32% unknown 200-300 ppm 200 ppm unknown unknown unknown

Uses Antifungal agent in breads, cake, Swiss cheeses Antifungal agent in cheeses, jellies, syrups, cakes Antifungal agent in margarine, cider, relishes, soft drinks Antifungal agent in breads Antimicrobial agent in cheeses, buttermilk, yogurt and pickled foods Antimicrobial agent in dried fruits, grapes, molasses Antibacterial agent in cured meats, fish Prevents microbial spoilage of meats, fish, etc. Prevents microbial spoilage of preserves, jams, syrups, jellies, etc. Prevents microbial spoilage of meats, fish, etc.

Chemotherapeutic agents: antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease. Examples: sulfonilamides, isoniazid, ethambutol, AZT, chloramphenicol. Note that the microbiologist's definition of a chemotherapeutic agent requires that the agent be used for antimicrobial purposes and so excludes synthetic agents used for therapy against diseases that are not of microbial origin. Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms. This is the microbiologist's definition. A more broadened definition of an antibiotic includes any chemical of natural origin (from any type of cell) which has the effect to kill or inhibit the growth of other types cells. Since most clinically-useful antibiotics are produced by microorganisms and are used to kill or inhibit infectious Bacteria, we will follow the classic definition. Antibiotics are low molecular-weight (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil. Most of these microorganisms form some type of a spore or other

dormant cell, and there is thought to be some relationship (besides temporal) between antibiotic production and the processes of sporulation. Among the molds, the notable antibiotic producers are Penicillium and Cephalosporium , which are the main source of the beta-lactam antibiotics (penicillin and its relatives). In the Bacteria, the Actinomycetes, notably Streptomyces species, produce a variety of types of antibiotics including the aminoglycosides (e.g. streptomycin), macrolides (e.g. erythromycin), and the tetracyclines. Endosporeforming Bacillus species produce polypeptide antibiotics such as polymyxin and bacitracin. The table below (Table 4) is a summary of the classes of antibiotics and their properties including their biological sources.
Table 4. Classes of antibiotics and their properties

Chemical class

Examples

Biological source

Spectrum (effective against)

Mode of action Inhibits steps in cell wall (peptidoglyc an) synthesis and murein assembly Inhibits steps in cell wall (peptidoglyc an) synthesis and murein assembly Suicide inhibitor of betalactamases Inhibits steps in cell wall (peptidoglyc an) synthesis and murein assembly Inhibits steps in cell wall (peptidoglyc an) synthesis

Beta-lactams (penicillins Penicillin G, and Cephalothin cephalosporin s)

Penicillium Gramnotatum and positive Cephalosporiu bacteria m species Grampositive and Gramnegative bacteria Grampositive and Streptomyces Gramclavuligerus negative bacteria Grampositive and Chromobacter Gramviolaceum negative bacteria Streptomyces Gramcattleya positive and Gramnegative

Semisynthetic Ampicillin, penicillin Amoxycillin

Clavulanic Acid

Clavamox is clavulanic acid plus amoxycillin

Monobactams Aztreonam

Carboxypene Imipenem ms

bacteria Grampositive and Aminoglycosid Streptomyces Streptomycin Grames griseus negative bacteria Grampositive and GramMicromonosp negative ora species bacteria esp. Pseudomona s Grampositive Streptomyces bacteria, orientales esp. Staphylococc us aureus Grampositive and GramStreptomyces negative lincolnensis bacteria esp. anaerobic Bacteroides

and murein assembly Inhibit translation (protein synthesis)

Gentamicin

Inhibit translation (protein synthesis) Inhibits steps in murein (peptidoglyc an) biosynthesis and assembly Inhibits translation (protein synthesis)

Glycopeptides Vancomycin

Lincomycins

Clindamycin

Macrolides

Grampositive bacteria, GramStreptomyces negative Erythromycin erythreus bacteria not enterics, Neisseria, Legionella, Mycoplasma Polymyxin Bacitracin Bacillus polymyxa Bacillus subtilis Gramnegative bacteria Grampositive

Inhibits translation (protein synthesis)

Polypeptides

Damages cytoplasmic membranes Inhibits steps in murein

bacteria

(peptidoglyc an) biosynthesis and assembly Inactivate membranes containing sterols Inactivate membranes containing sterols

Polyenes

Streptomyces Amphotericin Fungi nodosus

Nystatin

Streptomyces Fungi noursei (Candida)

Rifamycins

Rifampicin

Grampositive and Inhibits Gramtranscription Streptomyces negative (eubacterial mediterranei bacteria, RNA Mycobacteriu polymerase) m tuberculosis Grampositive and Streptomyces Gramspecies negative bacteria, Rickettsias Grampositive and Gramnegative bacteria, Rickettsias Ehrlichia, Borellia Inhibit translation (protein synthesis)

Tetracyclines Tetracycline

Semisynthetic Doxycycline tetracycline

Inhibit translation (protein synthesis)

Grampositive and Chlorampheni Chlorampheni Streptomyces Gramcol col venezuelae negative bacteria

Inhibits translation (protein synthesis)

Antimicrobial Agents Used in the Treatment of Infectious Disease

The modern era of antimicrobial chemotherapy began in 1929 with Fleming's discovery of the powerful bactericidal substance penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several other antibiotics were in clinical usage. The most important property of a clinically-useful antimicrobial agent, especially from the patient's point of view, is its selective toxicity, i.e., that the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the animal taking the drug This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy. Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic are is expressed as its spectrum of action. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum . If effective mainly against Gram-positive or Gramnegative bacteria, they are narrow spectrum . If effective against a single organism or disease, they are referred to as limited spectrum.